Neisseria gonorrhoeae is the causative agent for the sexually transmitted disease gonorrhea and was responsible for over 350,000 infections in the U.S. in 2007. The steady and inexorable increase of resistance in this organism toward multiple classes of antibiotics has severely limited treatment options for gonococcal infections and, after the recent withdrawal of fluoroquinolones, the expanded-spectrum cephalosporin ceftriaxone is now the only single-dose treatment recommended by the CDC in the U.S. Unfortunately, new strains of N. gonorrhoeae have emerged that exhibit resistance to cefixime and ceftriaxone, and treatment failures are now being reported. This precarious position endangers public health and demands a better understanding of antibiotic resistance at the molecular level, as well as strategies to develop new antimicrobials directed against N. gonorrhoeae. This renewal application will address this need by investigating two enzymes of N. gonorrhoeae involved in peptidoglycan synthesis. One is penicillin-binding protein 2 (PBP 2), a transpeptidase that forms peptide cross-links during the latter stages of cell wall synthesis, and the clinical target for ?-lactam antibiotics directed against this organism. Cephalosporin-resistant strains of N. gonorrhoeae harbor mutations in PBP 2 and a key goal is to determine the structural mechanisms that lower reactivity of PBP 2 with these antibiotics. We will also apply NMR relaxation methods to test the hypothesis that the molecular mechanism governing penicillin and cephalosporin resistance mediated by PBP 2 involves dynamic states of the protein. In recognition that other enzymes involved in peptidoglycan metabolism are potential targets for antimicrobials, we will also investigate N-acetylmuramyl-L-alanine amidase (AmiC), an autolysin that is required for proper cell division of N. gonorrhoeae. We have discovered that this enzyme exhibits autolytic activity in its N-terminal domain in addition to its known amidase activity in the C-terminal domain and therefore is a bifunctional autolysin. To understand the functional role of AmiC in peptidoglycan breakdown, but also to pave the way for drug discovery against its two active sites, we will obtain essential structural and biochemical information for AmiC.